In various fields ranging from fundamental biology to clinical diagnostic and public health surveillance, the specific and accurate quantification of proteins in complex biological samples remains a recurrent and challenging problem. For many protein biomarkers, this problem has been solved by immunological techniques. However, the success of immunological approaches relies on the heavy duty production and validation of high specificity and high affinity antibodies. Although recent efforts are being made to design antibodies arrays [20], the adaptation of immunological methods to multiplexed analyses remains limited. Indeed, the simultaneous optimization of several protein assays is hardly ever possible [21]. Alternatively, the power of MS-based proteomics can be harnessed to allow proteome-wide quantifications.
Mass spectrometry (MS) has greatly contributed to the maturation of proteomics [1]. It is now possible to characterize hundreds of proteins in an hour time frame and compare protein abundances in pairs of samples. The next frontier lies in accurate absolute quantitation. Although label-free spectral counting approaches [2, 3] are attracting considerable interest, robust absolute quantitative methodologies typically rely on the isotope dilution principle [4], in which internal standardization is achieved with isotope-labeled homologs of specific proteolytic peptides from the target protein(s) [5, 6]. The Absolute Quantitation (AQUA) peptide strategy uses chemically synthesized isotope-labeled peptides which are spiked into the samples in known quantities before MS-analysis [5-8]. The commercial availability of highly pure synthetic isotope-labeled peptides renders the AQUA peptide strategy very attractive. This methodology has been successfully used to quantify neuropeptides [23] or protein phosphorylations with phosphopeptides standards [5-7]. However, individual chemical synthesis, purification and quantification of isotope-labeled peptides make AQUA quantifications rather expensive. For this reason, proteins of interest are often quantified with a single AQUA peptide [24, 25].
Recently, the synthesis and metabolic labeling of an artificial concatemer of standard peptides (QCAT), which can be spiked into the samples before trypsin digestion, was introduced to extend the number of quantified proteins [9, 10]. QCAT and related polySIS polyproteins were developed as a smart intermediate strategy for multiplex absolute quantification of proteins. QCAT constructions allow the parallel production and quantification of several (up to 100) peptides in a single experiment. Several marker peptides representing a single protein can be included. Once conceived, a QCAT gene can easily be used for repeated production of unlimited amounts of isotope-labeled peptide standards. Interestingly, protein expression enables the synthesis of peptides difficult to produce by chemical methods such as peptides longer than 15 residues or peptides containing chemically reactive residues. According to Beynon et al [9], QCAT proteins should be especially suited for the assessment of stoichiometric ratios proteins within complexes.
The AQUA and QCAT strategies take advantage of identical chromatographic properties of an isotope labeled peptide and its unlabeled equivalent in the reverse phase chromatography step of LC-MS analyses.
Although AQUA and QCAT approaches have significantly advanced the quantitative measurement of proteins in biological samples, we discovered that the use of such standards can lead to severe biases. Calibration with AQUA peptides and QCAT constructs suffer from the following limitations: (i) a failure to take into account the actual efficiency of the proteolysis step required before MS analysis; (ii) an incompatibility with sample prefractionation which is often necessary when dealing with biological samples [11]; (iii) a poor protein sequence coverage, limiting the statistical reliability of the quantification.
Thus there is still an existing need to develop an accurate method for absolute quantification of polypeptides.